The process of copying information contained in the DNA of an organism into RNA by RNA polymerases is a crucial step in the biological expression of this information. Consequently, this is an important junction at which control of gene expression can be exerted. I propose to study a well characterized system, the interaction of Escherichia coli RNA polymerase with its specific functional sites on DNA (promoters), and the control of the process by the cI protein of Phage Lamda. I will focus on two regions of DNA with which the RNA polymerase makes contact when bound to a promoter, and the spacer DNA separating them. The interactions of RNA polymerase with each of the two regions will be studied separately. The effect of altering the relative rotational orientation of the two DNA regions on promoter functioning will be explored by modifications of the sequence of the spacer DNA rather than changing its length. These modifications will be introduced using newly developed rapid methods for chemical DNA synthesis, which allows for great versatility in manipulating DNA sequences. Cloning of the synthesized DNA will aid its manipulation and enable the testing of the modified promoters in vivo. A variety of biochemical and physical techniques will be used to determine the binding affinity of RNA polymerase for the constructed DNA substrates, the extend of distortion of spacer DNA by bound RNA polymerase, and the in vitro and in vivo functioning of the altered promoters. Many of the same promoter variants will be used to characterize in detail the mechanism of action of the weak phage promoters by the phage encoded cI protein. The intended research should significantly further our understanding of those determinants on the DNA of weak promoters which allow their activation by controlling proteins, as well as of those determinants which necessitate such an activation. These studies will provide the foundation to explore the regulation of RNA polymerase III catalyzed transcription in eukaryotic organisms.